Scleropodium is a genus of six species in the Brachytheciaceae. Although they are common in north temperate zones, they have not received monographic treatment in over a century. The aims of this study were to test species circumscriptions within the genus with molecular data, complete a thorough global taxonomic treatment of the genus, and to quantitatively investigate the ecological preferences of the species.

A molecular phylogenetic study was conducted using 104 individuals spanning the range of morphological variation and the geographic extent of the genus. Maximum Parsimony and Bayesian phylogenetic analyses and a statistical parsimony network analysis of ITS and the chloroplast rps4, bsbA2 and trnG regions were performed. Although slight differences were found among analyses, there were six clear molecular groups. Five of these corresponded directly to the species Scleropodium californicum, S. cespitans, S. julaceum, S. obtusifolium and S. touretii. The sixth species, S. occidentale, is new to science and is described here. It is similar in ecology and morphology to S. obtusifolium, but has several diagnostic features in both molecular markers and morphological characters. Molecular data also revealed that S. australe, previously recognized as an endemic species in Tasmania, is nested in phylogenetic analyses within European S. touretii.

For the taxonomic treatment, specimens from across the global distribution of the genus were obtained and examined. Earlier taxonomic problems are resolved and new determinations of many herbarium specimens resulted in substantial changes in the known geographic distributions of the species. Nomenclatural problems are addressed, including lectotypification of S. cespitans, neotypification of S. touretii, and snynonymization of S. colpophyllum with S. cespitans and S. australe with S. touretii. Two Korean species, S. brachyphyllum and S. coreense, are excluded from Scleropodium.

A study of niche differences among five of the six species was conducted in the coast ranges of California. The study employed twenty-eight100m2 plots to investigate mesosite and microsite differences among species. Differences could be found in all species pairs except for S. obtusifolium and S. occidentale, both of which are restricted to seasonal drainages. Environmental factors that were important at broad spatial scales included slope and tree canopy cover. Finer scale differences at the scale of individual moss patches were found in distance of the moss patches from drainages, substrate type and shade. Although niche differences were observed and quantified, niche overlap was extensive and mixed moss patches with two species growing intertwined were common. Niche differences among sexes within species was also investigated, but results were mostly not significant. In the more common species, there were trends toward female sex ratio bias and restriction of male individuals to more mesic habitats, but these results were mostly confounded by numbers non-expressing individuals.

Leaf surfaces of tropical vascular plants provide homes for diverse groups of organisms, including epiphyllous (leaf-colonizing) bryophytes. Each leaf harbors a temporally and spatially discrete community of organisms, providing an excellent system for answering some of the most fundamental questions in ecology and evolution. In this dissertation, I investigated two main aspects of epiphyllous bryophyte biology: 1) adaptive traits of bryophytes to living on the leaf surface, and 2) community assembly of epiphyllous bryophytes in space (between-hosts) and time (succession).

For the first part, I used published trait data and phylogeny of liverworts in family Lejeuneaceae to demonstrate that only the production of asexual propagules appeared to evolve in response to living on the leaf surface, while other hypothesized traits did not have correlated evolution with epiphylly. The second portion dealt with the assembly of communities among different host types. In this part, I identified communities of epiphyllous bryophytes from the same set of three hosts across the gradient of water deficit fluctuation, as represented by the daily range of vapor pressure deficit (VPD) on the island Moorea, French Polynesia. Dissimilarity of epiphyllous communities among host types (beta diversity) increased with the increasing VPD range, suggesting that assembly of bryophyte community may depend on microclimate fluctuation. In the last portion of this dissertation, I investigated temporal changes of epiphyllous communities on Inocarpus fagifer, using chronosequence and long-term observation of tagged leaves in the field. Data from both approaches showed significant changes in abundance, species richness, and composition over time. These successional changes, however, did not follow any single trajectory, resulting in divergent communities among leaves of older age. Together, the results from this dissertation work improve our understanding of fundamental biology of epiphyllous bryophytes and allow future researchers to use this system to answer broader questions in ecology and evolution.

An environmental stressor, UV radiation is a potentially important selective factor in ecosystems with high solar exposure. Natural solar radiation can be damaging to plants, including to important components of plant metabolism. In addition to protecting tissues from dangerous levels of radiation, maintaining access to sufficient water is also an important problem for terrestrial organisms to solve. Common in drylands, the moss genus Syntrichia contains some of the most desiccation-tolerant plants known. Desiccation tolerance (DT), defined as the ability to equilibrate to dry air and resume normal metabolic activity after rehydration, allows organisms to survive dry periods and limit metabolic activity to periods of moisture availability. These resilient mosses can lose most of their cellular water, remain quiescent for decades, and begin photosynthesizing and recovery immediately upon rehydration.

In this dissertation, I explore molecular and physiological responses of two Syntrichia species to UV radiation in a variety of field, laboratory, and combined experiments to gain insight on the adaptive mechanisms allowing these organisms to thrive in dryland habitats. The first chapter is an eco-physiology study that focuses on the effects of natural levels of UV radiation on photosynthesis. The second and third chapters are controlled laboratory experiments that explore plasticity in the response to different levels of UV radiation and, in the third, how the response might interact with desiccation.

In Chapter 1, I used an in-situ field experiment to uncover the effects of natural and reduced levels of UV on maximum PSII quantum efficiency (Fv/Fm), relative abundance of photosynthetic pigments and antioxidants, and on the transcriptome in the desiccation-tolerant desert moss Syntrichia caninervis. I tested the hypotheses that: (1) natural S. caninervis plants undergo sustained thermal quenching of light (non-photochemical quenching; NPQ) while desiccated and after rehydration, (2) a reduction of UV will result in improved recovery of Fv/Fm, but (3) one year of UV removal will de-harden plants and increase vulnerability UV damage to photosynthetic efficiency. All field-collected plants had extremely low Fv/Fm after initial rehydration but recovered over eight days in lab-simulated winter conditions. UV-filtered plants had lower Fv/Fm during recovery, higher concentrations of photoprotective pigments and antioxidants such as zeaxanthin and tocopherols, and lower concentrations neoxanthin and chlorophyll b than plants exposed to near natural UV levels. Natural S. caninervis undergoes sustained NPQ that takes days to relax and for efficient photosynthesis to resume. Reduction of solar UV radiation adversely effects recovery of Fv/Fm following rehydration.

In Chapter 2, I investigated the genetic underpinnings to acute broadband UV exposure in S. caninervis and S. ruralis, using a comparative transcriptomics approach. In particular, I aimed to uncover whether UV protection is physiologically plastic and induced by UV exposure with the following questions: (1) what is the timeline of changes in the transcriptome with acute UV exposure in these two species, (2) what genes are involved in the acute UV response, and (3) how do the two species differ in their transcriptomic response to UV. There was no strong transcriptomic response with 10 minutes of UV exposure in either S. ruralis or S. caninervis and relatively small number of significant genes at 30 minutes, suggesting a physiologically constitutive, rather than plastic or acclimated, protection to UV radiation. Yet, there was remarkable differences between the two species after 30 minutes, including nearly twice as many significant genes (including two early light-inducible protein genes) for S. caninervis than for S. ruralis. Many significant genes in both species were involved in oxidation-reduction, suggesting oxidative stress. Late embryogenesis abundant genes were also involved in both species in response to UV, possibly suggesting their role in UV tolerance or as evidence of cross-talk for desiccation tolerance. Taken together, the results of this study suggest potential UV-induced responses that might have roles outside of desiccation tolerance, and that the response to UV is different in these two species, perhaps a reflection of adaption to different environmental conditions.

In Chapter 3, I conducted a fully factorial experiment comparing the separate and combined effects of two levels of UV radiation and a desiccation treatment in S. ruralis and S. caninervis to uncover the nature of correlation between DT and UV tolerance. When exposure to one type of stressor confers or enhances protection to another type it can be due to either “cross-tolerance” (overlap in the mechanism of protection for two or more types of stressors) or “cross-talk” (when there is overlap in signaling pathways in response to different stressors but separate mechanisms of protection). I tested the following hypotheses: (1) There is cross-talk in the genetic underpinnings of the UV and desiccation response pathways for S. caninervis. (2) S. ruralis will instead show a pattern of cross-tolerance with UV and desiccation stresses, evidenced by distinct transcriptomic responses to the two stressors but overlap in metabolomic responses. I found evidence of both cross-talk and cross tolerance in both species, but with nuanced differences in their response to the individual and combined stresses. In particular, I found support for the hypothesis of cross-talk in the UV and desiccation response pathway for S. caninervis, evidenced by shared transcriptomic response to the two stressors with no significant interaction. I also found key transcripts and metabolites that shed light on the mechanisms of tolerance to these two stressors, both individually and combined. Notably, phenolics were involved in the UV response at both the transcriptomic and metabolomic levels, though with differences between species and interactions with desiccation, indicating potential for a UV sunscreen and for cross-talk and cross-tolerance in these species. Furthermore, there were candidate UVT genes and metabolites that were not induced by UV in S. caninervis but were in S. ruralis, supporting the hypothesis that S. ruralis has a more plastic, acclimatable UV response than S. caninervis, and that these differences are predictable by their unique interaction with these stressors as poikilohydric organisms. The genetic and metabolomic findings of this study can be directly used to test hypotheses and further elucidate mechanisms of UV protection and VDT in these stress-tolerant plants and a greater understanding of mechanism can lead to development of engineering technologies to improve crop plants’ ability to withstand combined stresses.

In the midst of global climate change, it is crucial to understand the spatial patterns of biodiversity and its underlying ecological and evolutionary mechanisms, including neglected groups in the tree of life such as bryophytes (liverworts, mosses, and hornworts). These lineages share some important characters with tracheophytes (e.g., embryonic development and elaboration of the sporophyte generation), but also, they have retained characters from their freshwater algal ancestors (e.g., desiccation tolerance, poikilohydry, and dominant gametophyte). Because of their unique biology, they are ideally suited for addressing questions in evolutionary biology, functional morphology, and biodiversity.

This Dissertation consists of three independent but interconnected topics using mosses as a study system. In Chapter 1, I aim to understand the patterns of diversity and community structure of mosses along elevation and moisture gradients in Central Chile. Chapter 2 addresses the evolutionary history of the dryland moss Syntrichia Brid. Chapter 3 focuses on functional morphology in relation to external water-conduction in Syntrichia.

The goal of Chapter 1 was to investigate the diversity of mosses along an elevational gradient in Central Chile. I used phylogenetic approaches (phylogenetic diversity, relative phylogenetic diversity, and phylogenetic turnover) to measure the diversity and community structure of 25 sites along an elevation gradient in Central Chile. To understand the composition of moss communities according to soil moisture, each site was subdivided into three subsites arranged perpendicularly to a water source (e.g., creek) ranging from completely dry to fully moist. The phylogenetic pattern suggested that environmental filtering was responsible for the co-occurrence of closely related taxa at low and high elevations, and that competition had a minimal effect on community assembly. Phylogenetic turnover revealed changes in community composition along elevation and soil moisture.

In Chapter 2, I aimed to investigate the evolutionary, biogeographic history, and trait evolution of Syntrichia, through the most comprehensive phylogenetic analysis performed up-to-date. A combined phylogeny based on a hundred nuclear loci plus a robust morphological matrix suggested that Syntrichia is monophyletic with 10 major subclades. It is very likely Syntrichia originated in South America in the early Eocene and dispersed to other landmasses via dispersal. The genus experienced major diversification events in South America and Northern Hemisphere, the areas with the current greatest species diversity. Habitat preferences (e.g., soil, rocks, and trees) might be associated with evolutionary changes in water-related traits.

Chapter 3 explored how the moss Syntrichia absorbs, conducts, and retains water externally, a process known as ectohydry. I used a diverse range of microscopic techniques for observing anatomy as well as experimental approaches to understand the rate of conduction and dehydration. I propose a new framework for studying ectohydric capabilities that include three spatial scales (cell anatomy, stem architecture, and whole clump) and timing to become fully hydrated. There was a trade-off between speed of conduction and holding capacity that could be associated with the life history of each species.

This dissertation is a contribution to the understanding of several fundamental questions in the biology of mosses. This work will provide the basis for future research in the field of evolution, ecology, and functional morphology.

Abstract

Early Trait Evolution in Liverworts (Marchantiophyta), with an emphasis on Oil Bodies and Paleozoic Taxa

By

Susan Lorraine Tremblay

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Brent D. Mishler, Chair

Liverworts (Marchantiophyta) are a diverse and phylogenetically important group of land plants (Embryophytes). They diverged early on from the rest of the land plants, but few fossils exist from the Paleozoic or Mesozoic and those that do exist generally possess only a few vegetative characters. By the Cenozoic, when liverwort fossils become more common, modern groups had evolved and the fossils can be easily placed. Consequently little is known about early character evolution in liverworts. Recently, studies based on molecular data have resolved the liverwort phylogeny, and together with new fossil discoveries they offer an opportunity to reassess the early evolution of liverworts.

One of the most important liverwort characters are the unique organelles found in most of their cells called oil bodies. Considered a synapomorphy of liverworts, oil bodies are bound by a biological membrane and synthesize and sequester a wide variety of terpenoids and aromatic compounds, yet their exact role in the cell is not known. They can easily be observed through a light microscopy and are taxonomically informative. The first chapter of this thesis reviews what is known about oil bodies from a phylogenetic perspective. Structure and chemistry of oil bodies is reviewed, with special attention to assignment of oil body 'types' to groups based on the recent classification of liverworts.

Hypotheses of oil body evolution generally assume an ancestor with a single type of oil body, with the present diversity of oil bodies having evolved from the ancestral type. As documented in this review, similar variation in oil body types occurs in distantly related clades across the newly resolve phylogeny, including the earliest diverging groups, suggesting that the most recent common ancestor of the group may have been polymorphic for oil body types. Some liverwort taxa have secondarily lost oil bodies. Comparing these taxa to their sister groups could offer insight into oil body function. Some experimental evidence supports the anti-herbivory hypothesis of oil body function. Hypotheses of oil body evolution and oil body function need to be tested using both systematic and ecological methods.

In some groups oil bodies occur only in special, scattered cells that contain one large oil body. In my second chapter I test the hypothesized homology of the dark scattered cells of some Paleozoic liverwort fossils with the specialized oil body cells of some modern liverwort taxa. Fresh fossils of the oldest known liverwort, the recently discovered Metzgeriothallus sharonae Hernick, were collected and isolated from the matrix and their cells were compared to those of modern taxa using light microscopy, scanning electron microscopy, and fluorescence microscopy. Frequency of occurrence and area coverage of the dark cells were compared to several modern taxa. All results support the homology of the fossil dark cells and modern oil body cells, providing an important new character for use in assigning fossils to the liverworts and potentially to specific groups. It will also help elucidate the plesiomorphic state of liverwort oil bodies. The results showed that the dark cells of M. sharonae are more numerous near thallus margins providing circumstantial evidence for the anti-herbivory hypothesis of their function.

Metzgeriothallus sharonae is spectacularly preserved in situ and the acid maceration revealed complex branching previously unknown in Paleozoic liverworts. While M. sharonae was the dominant plant in the assemblage, similarly well-preserved organic remains of dozens of co-existing organisms were also recovered. In the third chapter of this thesis I describe these new features and the community these ancient liverworts were a dominant member of. The dimorphic structure of the plants, broad, dichotomizing thalli alternating with narrower, twisting thalli that have many intercalary branches suggests the plants were growing both above and below the surface of and along banks of shallow pools. Metzgeriothallus sharonae is the only Paleozoic thalloid liverwort to exhibit intercalary branching as well as ventral branching at the thallus apex. The Cairo Quarry liverwort lens represents a conservation lagerstatte, a miniature 'frozen' freshwater ecosystem that is the only known liverwort dominated community from the Paleozoic.

Regeneration is common amongst all classes of echinoderms and is easily studied in the laboratory with abundant and tractable ophiuroid specimens. However, ophiuroid regeneration rates, both between species and within the same species, can exhibit uncommonly high variation even under identical laboratory conditions. Eighty-four Ophiocoma scolopendrina (Lamarck 1816), a brittle star which extends its arms out into the water column to suspension feed, were collected from the upper-intertidal zone of rocky beaches on the island of Moorea, French Polynesia for laboratory regeneration experiments. To determine the effects of position of autotomy and feeding level on regenerated length and differentiation of new biomass, two treatments were performed: an amputation treatment either 5 mm from the central disk or 30 mm from the distal tip; and a feeding treatment of high-food or low-food. Additionally, a field survey was conducted to assess predation pressure and autotomy of O. scolopendrina in upper-intertidal zones. This study demonstrated that O. scolopendrina are well adapted to the high levels of sub-lethal predation at the arm tips by quickly resuming the ability to detect predators via photo and chemoreception. The results of this investigation can help to better inform calculations of energy cycling in ecosystems and has potential applications for further research on the connections between ophiuroid life-history and predation pressure on variable regeneration rates.

The genus Artemisia

Biogeography and chemosystematics in a phylogenetic context (Phylochemistry)

By Christopher Rollin Hobbs

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Bruce G. Baldwin, Chair

Artemisia is the most diverse genus in tribe Anthemideae, consisting of 350 to 500 species (Hillebrand, 1888; Vallès & McArthur, 2001; Vallès et al., 2003). These wind-pollinated herbaceous and shrubby taxa, including mugworts, sagebrushes, and wormwoods, occur widely in the Northern Hemisphere and sparingly in southern South America and sub- Saharan Africa. I studied the biogeography of the genus with an emphasis on the three Hawaiian taxa in the context of world-wide distribution.

I sought to determine whether Hawaiian Artemisia (Compositae-Anthemideae), with lowland and subalpine species, represents such an example by investigating the origin and relationships of the Hawaiian taxa.

Molecular phylogenetic analyses of Hawaiian Artemisia were conducted using nuclear ribosomal transcribed spacers, chloroplast DNA intergenic spacers, and morphology. Timing of divergence events associated with inferred dispersals was estimated with calibration from fossil pollen records. Historical biogeographic analyses based on molecular trees and ecological modeling of distributions of extant taxa were used to aid interpretation of geographic and habitat shifts associated with diversification and long-distance dispersal.

The findings indicate that the Hawaiian-endemic species (A. australis, A. kauaiensis, and A. mauiensis) constitute a clade that is sister to southeast Asian A. chinensis, which, like the Hawaiian endemics, has ribbed fruit walls and, unlike other members of Artemisia: except A. kauaiensis, has a distinct pappus, often associated with dispersal ability in Compositae. The clade encompassing A. chinensis and Hawaiian Artemisia was resolved to be most likely of Asian origin.

An ecological shift in Hawaiian Artemisia from tropical coastal habitats to drier and colder subalpine slopes is consistent with evidence from recent studies by Tkach and others, and data reported here, for repeated colonisation of the arctic by diverse lineages of Artemisia. Artemisia appears to be prone to such anticlimatic ecological shifts, which may explain this exceptional example of an ancestrally lowland tropical lineage in the Hawaiian high-montane flora.

Since the genus Artemisia has an abundance of secondary metabolites (SM), from a variety of distinct biosynthetic pathways, I sought to determine if these compounds had biogeographical signal, and were more influenced by environment, or mostly under genetic control and conserved. The odor compounds, or volatile organic constituents (VOCs), with up to 200 possible in one species was characterized by gas chromatograph/mass spectroscopy, and are a strong characteristic of the genus. Based on their odors, they seem to be distinct and varied within individual species.

First I sought to determine if compounds from all major constituent classes (alkaloids, terpenes, fatty acids, phenolic compounds) were under environmental or genetic control. The use of SM for solving taxonomic problems is called chemotaxonomy or chemosystematics. Use of SM in systematics, by tradition called chemosystematics, has been extensive for more than a half century and predates macromolecular approaches.

A comprehensive search of the chemosystematics literature was undertaken, and a previously published dataset of 24 leaf fatty acids for 123 taxa from all major gymnosperm clades (cycads, ginkgos, gnetophytes, and conifers) was compared with trees from rbcL chloroplast sequence data for the same taxa using comparative analyses in R to determine phylogenetic signal and to evaluate the mode of evolution of the chemical traits. Strong evolutionary constraint of the fatty acid data adds weight to the idea that fatty acids are more suitable for use as characters in phylogenetic analyses than non-structural secondary compounds. The relative benefits and drawbacks of chemical data are briefly discussed, along with potential problems in their collection, extraction, analysis, and interpretation.

Volatile organic constituents are prominent in the genus Artemisia, and many of these volatile compounds such as thujone and 1,8-cineol, are associated with defensive behavior such as anti- herbivory, anti-encroachment, and antimicrobial effects in plants. I sought to test the hypothesis that these toxic compounds might be lost in island settings over evolutionary time because of a demonstrable lack of herbivores and other encroaching species within the time frame of early colonization. I show show a dramatic example of island tameness in plants in Artemisia. In the 74 species sampled across the genus, 10 abundant and widely-studied, toxic terpene compounds that are known to inhibit seed germination, root development, and herbivore feeding9 demonstrate repeated loss, in two separate island lineages in the Hawaiian and Canary Islands. This is the first phylogeny-based study demonstrating this in plants, whereas island tameness has been widely discussed in animals, and well-known examples such as the dodo of Mauritius, Galápagos island iguanas are common knowledge. The tameness of the Falkland Island wolf, was noted by Darwin.

Simple visible-light digital cameras offer a potential for expanded forms of plant ecological research. The moss Tortula princeps undergoes changes in reflected visible light during cycles of drying and hydrating in the field, and the MossCam project has collected digital images of T. princeps at least daily since 2003. Laboratory studies can be used to calibrate these images to indicate field physiological conditions. Drying the moss 6 d in the laboratory resulted in a decrease of net CO2 uptake to near 0; recovery after rewetting occurred within 10 min. The difference in reflectance between hydrated and dry T. princeps was maximal ca. 550 nm, and maximal net CO2 uptake was linearly related to the green : red ratio of laboratory images when net CO2 uptake was positive. Using the green : red ratio of field images and otherwise assuming ideal conditions, the total carbon gain for a 6-d period around a 1.3-mm rain event was ca. 208 mmol CO2 /m2, equivalent to 69 d of respiration under dry conditions. Using a visible-light digital camera with micrometeorological data and laboratory-based gas exchange measurements, T. princeps can be used as a model species for simple field estimations of photosynthesis, carbon gain, and phenological events.

Graham, E.A., M.P. Hamilton, B.D. Mishler, P.W. Rundel & M.H. Hansen. 2006. Use of a networked digital camera to estimate net CO2 uptake of a desiccation tolerant moss. International Journal of Plant Sciences 167:751-758.